Glial Cell Line-derived Neurotrophic Factor
Glial cell line-derived neurotophic factor (GDNF) was initially isolated and cloned from rat B49 cells as a potent neurotrophic factor that enhances survival of midbrain dopaminergic neurons (Lin et al., Science, 260, 1130-1132, 1993). Recent studies have indicated that this molecule exhibits a variety of other biological activities, having effects on several types of neurons from both the central and peripheral nervous systems. In the central nervous system (CNS), GDNF has been shown to prevent the axotomy-induced death of mammalian facial and spinal cord motor neurons (Li et al., Proceedings Of The National Academy Of Sciences, U.S.A., 92, 9771-9775, 1995; Oppenheim et al., Nature, 373, 344-346, 1995; Yan et al., Nature, 373, 341-344, 1995; Henderson et al., Science, 266, 1062-1064, 1994; Zurn et al., Neuroreport, 6, 113-118, 1994), and to rescue developing avian motor neurons from natural programmed cell death (Oppenheim et al., 1995 supra). Local administration of GDNF has been shown to protect nigral dopaminergic neurons from axotomy-induced (Kearns and Gash, Brain Research, 672, 104-111, 1995; Beck et al., Nature, 373, 339-341, 1995) or neurotoxin-induced degeneration (Sauer et al., Proceedings Of The National Academy Of Sciences U.S.A., 92, 8935-8939, 1995; Tomac et al., Nature, 373, 335-339, 1995). In addition, local administration of GDNF has been shown to induce sprouting from dopaminergic neurons, increase levels of dopamine, noradrenaline, and serotonin, and improve motor behavior (Tomac et al., 1995 supra).
More recently, GDNF has been reported to be a potential trophic factor for brain noradrenergic neurons and Purkinje cells. Grafting of fibroblasts ectopically expressing GDNF prevented 6-hydroxydopamine-induced degeneration and promoted the phenotype of adult noradrenergic neurons in vivo (Arenas et al., Neuron, 15, 1465-1473, 1995), while exogeneously applied GDNF effectively promoted survival and morphological differentiation of embryonic Purkinje cells in vitro (Mount et al., Proceedings Of The National Academy Of Sciences U.S.A., 92, 9092-9096, 1995). In the peripheral nervous system, GDNF has been shown to promote the survival of neurons in nodose, ciliary, and sympathetic ganglia, as well as small populations of embryonic sensory neurons in dorsal root ganglia (DRG) and trigeminal ganglia (Trupp et al., Journal Of Cell Biology, 130, 137-148, 1995; Ebendal et al., Journal Of Neuroscience Research, 40, 276-284, 1995; Oppenheim et al., 1995 supra; Yan et al., 1995 supra; Henderson et al., 1994 supra). GDNF has also been reported to enhance the expression of vasoactive intestinal peptide and preprotachykinin-A mRNA in cultured superior cervical ganglion (SCG) neurons, and thus effects the phenotype of SCG neurons and induces bundle-like sprouting (Trupp et al., 1995 supra).
Expression of GDNF has been observed in a number of different cell types and structures of the nervous system. In the CNS, GDNF mRNA expression has been observed by reverse transcriptase polymerase chain reaction (RT-PCR) in both developing and adult rat striatum, the major target of nigral dopaminergic innervation, and widely in other regions, including hippocampus, cortex, thalamus, septum, cerebellum, spinal cord, and medulla oblongata (Arenas et al., supra 1995; Poulsen et al., Neuron, 13, 1245-1252, 1994; Springer et al., Experimental Neurology, 127, 167-170, 1994; Stroemberg et al., Experimental Neurology, 124, 401-412, 1993; Schaar et al., Experimental Neurology, 124, 368-371, 1993). In human, GDNF transcripts have also been detected in striatum, with highest level in the caudate and lower levels in the putamen. Detectable levels are also found in hippocampus, cortex, and spinal cord, but not in cerebellum (Schaar et al., Experimental Neurology, 130, 387-393, 1994; Springer et al., 1994 supra). In the periphery, GDNF mRNA expression has been reported in DRG and SCG of postnatal day I rats, sciatic nerve, and primary cultures of neonatal Schwann cells (Trupp et al., 1995 supra; Hoffer et al., Neuroscience Letters, 182, 107-111, 1994; Henderson et al., 1994 supra; Springer et al., 1994 supra). In addition, recent studies have shown that GDNF transcripts are also widely expressed in peripheral non-neuronal organs, including postnatal testis and kidney, embryonic whisker pad, stomach, and skin. Expression can be detected at lower levels in embryonic muscle, adrenal gland and limb bud, and in postnatal lung, liver and ovary (Trupp et al., 1995 supra; Henderson et al., 1994 supra). So far, however, the biological significance of the non-neuronal expression of GDNF is not clear.
A neurotrophic factor refered to as “neurturin” is described in Nature 384(5):467-470, 1996. Detailed descriptions of the preparation and characterization of GDNF protein products may be found in U.S. patent application Ser. No. 08/182,183 filed May 23, 1994, now abandoned, and its parent applications (also see PCT/US92/07888, WO 93/06116 filed Sep. 17, 1992 and European Patent Application No. 92921022.7, Publication No. EP 610 254) the disclosures of which are hereby incorporated by reference. Additional GDNF protein products are described in U.S. patent application Ser. No. 08/535,681 filed Sep. 28, 1995, now U.S. Pat. No. 6,184,200, the disclosure of which is hereby incorporated by reference. As used herein, the term “GDNF protein product” includes biologically active synthetic or recombinant GDNF proteins and analogs, as well as chemically modified derivatives thereof. GDNF analogs include deletion variants such as truncated GDNF proteins, as well as insertion and substitution variants of GDNF. Also included are GDNF proteins that are substantially homologous to the human GDNF protein.
GDNF Therapy
GDNF therapy is helpful in the treatment of nerve damage caused by conditions that compromise the survival and/or proper function of one or more types of nerve cells. Such nerve damage may occur from a wide variety of different causes. Nerve damage may occur to one or more types of nerve cells by: (1) physical injury, which causes the degeneration of the axonal processes and/or nerve cell bodies near the site of injury; (2) temporary or permanent cessation of blood flow to parts of the nervous system, as in stroke; (3) intentional or accidental exposure to neurotoxins, such as chemotherapeutic agents (e.g., cisplatinum) for the treatment of cancer or dideoxycytidine (ddC) for the treatment of AIDS; (4) chronic metabolic diseases, such as diabetes or renal dysfunction; or (5) neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis (ALS), which result from the degeneration of specific neuronal populations.
Several studies indicate that GDNF therapy is particularly helpful in the treatment of neurodegenerative conditions such as the degeneration of the dopaminergic neurons of the substantia nigra in Parkinson's disease. The only current treatments for Parkinson's disease are palliative, aiming at increasing dopamine levels in the striatum. The expected impact of GDNF therapy is not simply to produce an increase in the dopaminergic neurotransmission at the dopaminergic nerve terminals in the striatum (which will result in a relief of the symptoms), but also to slow down, or even stop, the progression of the degenerative processes and to repair the damaged nigrostriatal pathway and restore its function. GDNF may also be used in treating other forms of damage to or improper function of dopaminergic nerve cells in human patients. Such damage or malfunction may occur in schizophrenia and other forms of psychosis. The only current treatments for such conditions are symptomatic and require drugs which act upon dopamine receptors or dopamine uptake sites, consistent with the view that the improper functioning of the dopaminergic neurons which innervate these receptor-bearing neuronal populations may be involved in the disease process.
Receptor
A number of receptors which mediate binding and response to protein factors have been characterized and molecularly cloned, including receptors for insulin, platelet derived growth factor, epidermal growth factor and its relatives, the fibroblast growth factors, various interleukins, hematopoietic growth factors and ciliary neurotrophic factor (U.S. Pat. No. 5,426,177). Study results indicate that some receptors can bind to multiple (related) growth factors, while in other cases the same factor can bind and activate multiple (related) receptors (e.g., Lupu et al., Science, 249:1552-1555, 1990; Dionne et al., EMBO J., 9:2685-2692, 1990; Miki et al., Science, 251:72-75, 1991). Most receptors can broadly be characterized as having an extracellular portion or domain responsible for specifically binding a protein factor, a transmembrane domain which spans the cell membrane, and an intracellular domain that is often involved in initiating signal transduction upon binding of the protein factor to the receptor's extracellular portion. Although many receptors are comprised of a single polypeptide chain, other receptors apparently require two or more separate subunits in order to bind to their protein factor with high-affinity and to allow functional response following binding (e.g., Hempstead et al., Science, 243:373-375, 1989; Hibi et al., Cell, 63:1149-1157, 1990).
The extracellular and intracellular portions of a given receptor may share common structural motifs with the corresponding regions of other receptors, suggesting evolutionary and functional relationships between different receptors. These relationships can often be quite distant and may simply reflect the repeated use of certain general domain structures. For example, a variety of different receptors that bind unrelated factors make use of “immunoglobulin” domains in their extracellular portions, while other receptors utilize “cytokine receptor” domains in their factor-binding regions (e.g., Akira et al., The FASEB J., 4:2860-2867, 1990). A large number of receptors with distinct extracellular binding domains (which thus bind different factors) contain related intracytoplasmnic domains encoding tyrosine-specific protein kinases that are activated in response to factor binding (e.g., Ullrich and Schlessinger, Cell, 61:203-212, 1990). The mechanisms by which factor-binding “activates” the signal transduction process is poorly understood, even in the case of receptor tyrosine kinases. For other receptors, in which the intracellular domain encodes a domain of unknown function or in which the binding component associates with a second protein of unknown function (e.g., Hibi et al., Cell, 63:1149-1157, 1990), activation of signal transduction is not well characterized.
The mode of action of GDNF in vivo is not clearly elucidated in the art, in part due to the absence of information on a receptor for GDNF. Two groups have independently found that striatum injected [125I]-labeled GDNF can be retrogradely transported by dopaminergic neurons in the substantia nigra (Tomac et al., Proceedings Of The National Academy Of Sciences Of The United States Of America. 92, 8274-8278, 1995; Yan et al., 1995 supra). Retrograde transport of [125I]-GDNF by spinal cord motor neurons, DRG sensory neurons and neurons in the B layer of retina ganglia was also been observed. These retrograde transport phenomena can all be specifically inhibited by 100-fold or higher concentrations of unlabeled GDNF, suggesting a saturable, receptor-mediated transport process. In vitro, recombinant GDNF has been shown to enhance the survival and promote dopamine uptake of cultured dopaminergic neurons at very low concentrations. The observed half-maximal effective concentration (EC50) of GDNF on these neurons is 0.2 to 1.6 pM (Lin et al., 1993 supra). GDNF has also been shown to support the survival of dissociated motor neurons at low concentrations. The reported EC50 of GDNF on motor neurons, in a 5 to 10 fM range, is even lower than that on dopaminergic neurons (Henderson et al., 1994 supra).
Taken together, these observations indicate that receptor(s) for GDNF expressed in these cells have very high ligand binding affinities. Similar to members of the TGF-β family, the widely diversified tissue distribution and varied biological function of GDNF on different populations of cells suggest that different types of receptor(s) for GDNF or receptor complexes may exist. Saturation steady-state and competitive binding of [125I]-GDNF to E10 chick sympathetic neurons has shown that these neurons express GDNF binding sites differing from those observed in dopaminergic and motor neurons. The half maximal saturation concentration and the half-maximal inhibition concentration of GDNF on these binding sites is in the range of 1 to 5 nM (Trupp et al., 1995 supra). Similarly, the EC50 of GDNF in supporting the survival of sympathetic neurons from P1 rat SCG has also been reported to be in the nanomolar range (Trupp et al., 1995 supra).
To better understand the mechanism by which GDNF activates signal transduction to exert its affects on cells, it would be beneficial to identify the receptor(s) which mediate binding and response to this protein factor. It would also be beneficial for GDNF therapy to identify and make possible the production of accessory molecules which provide for or enhance GDNF signal transduction. Moreover, the identification of a protein receptor for GDNF would provide powerful applications in diagnostic uses, for example, as an aid in determining if individuals would benefit from GDNF protein therapy. Furthermore, the protein receptor for GDNF could be a key component in an assay for identifying additional molecules which bind to the receptor and result in desired biological activity.